1United States Geological Survey, Columbia, South Carolina, United States of America2United States Geological Survey, Troy, New York, United States of America3United States Geological Survey, Reston, Virginia, United States of America4United States Geological Survey, Mounds View, Minnesota, United States of America

Net fluxes (change between upstream and downstream margins)
for water, methyl mercury (MeHg), total mercury (THg), dissolved
organic carbon (DOC), and chloride (Cl) were assessed twice in
an Adirondack stream reach (Sixmile Brook, USA), to test the
hypothesized importance of wetland-stream hydraulic and chemical
gradients as fundamental controls on fluvial mercury (Hg) supply.
The 500 m study reach represented less than 4%of total upstream
basin area. During a snowmelt high-flow event in May 2009surface
water, DOC, and chloride fluxes increased by 7.1±1.3%, 8.0±1.3%,
and 9.0±1.3%, respectively, within the reach, demonstrating that the
adjacent wetlands are important sources of water and solutes to the
stream. However, shallow groundwater Hg concentrations lower than
in the surface water limited groundwater-surface water Hg exchange
and no significant changes in Hg (filtered MeHg and THg) fluxes were
observed within the reach despite the favorable hydraulic gradient.
In August 2009, the lack of significant wetland-stream hydraulic
gradient resulted in no net flux of water or solutes (MeHg, THg, DOC,
or Cl) within the reach. The results are consistent with the wetland-
Hg-source hypothesis and indicate that hydraulic and chemical
gradient (direction and magnitude) interactions are fundamental
controls on the supply of wetland Hg to the stream.

Introduction

Identifying methyl mercury (MeHg) source areas and
the controls on MeHg supply to streams is an environmental
priority due to widely-reported correlations between elevated
fluvial-MeHg concentrations and fish-Hg contamination
[1-3] , the leading cause of fish consumption advisories in
streams throughout the United States (US) [4] and Canada [5].
Strong correlations between wetland area and fluvial MeHg
concentrations [6-8] , higher dissolved MeHg concentrations [7,9-
11] and Hg methylation rates [7,12] in wetlands compared with
adjacent stream habitats, and strong wetland-stream hydrologic connectivity [13-16] support the hypothesis that wetland areas
are primary sources of fluvial MeHg in many systems [1,10-12,17-
19]. This wetland-MeHg-source hypothesis, in turn, predicts that
unfavorable wetland-stream hydraulic (water) and chemical
(MeHg) gradients may limit fluvial MeHg concentrations and
fluxes in the adjacent stream reach.

and concentration gradients were assessed in Sixmile Brook
during snowmelt and later during the growing season, to test the
hypothesis that unfavorable hydraulic and chemical gradients
(i.e., insignificant or from surface-water toward groundwater)
limit fluvial MeHg supply in this and in similar systems in the
Adirondacks region.

Material and Methods

Study Basins

The Upper Hudson River basin is in the Adirondack region of the Appalachian Highlands of New York [7,20,22,23], where
elevated biotic Hg concentrations are reported in lentic systems
[21,24-27]. The Sixmile Brook (18 km2 basin area) headwater
study basin has been described in detail elsewhere[7,20].
A500 m study reach was established between the upstream
confluence with Sixmile Tributary and a downstream hydraulic
control (bridge abutment)(Figure 1)[7,20]). The Coastal Plain
comparison reach in McTier Creek, South Carolina has been
described in detail elsewhere (Figure 1) [7,13,20]).

Out-of-channel (groundwater) piezometers (2.5 cm diameter
PVC) were installed on both banks with screened intervals 30-60
cm below land surface (Figure 1; Figures S1 and S2 in File S1). Only
data from 20 piezometers closest (3-10 m) to the stream channel
are discussed in this paper. In-streamhyporheic piezometers (4
each at top and bottom of reach; 1 cm diameters; 30 cm screened
intervals) were emplaced at the edge of water on each bank and
equidistant across the channel with the top of screen set at 20 cm
below the bed sediment/surface-water interface. Fine scale (1.5
cm minimum resolution) vertical solute gradients were assessed
at edge-of-water (margin) and center-of-channel (channel)at the
downstream reach boundary using two mini-
Piezometer (USGS
MINIPOINT) devices (seven 0.325 cm stainless steel tubes, 0.8 cm
screened interval) [28,29]. Each MINIPOINT device was installed
with sampling points at approximately 1.5, 4.5, 7.5, 12.5, 17.5, 30,
and 60 cm below the bed-sediment/surface water interface.

Sampling Event Hydrologic Setting

Lagrangian (following same average parcel of water
downstream through the study reach) surface-water flux
assessments (described in detail below) were initiatedin2009
during snowmelt in May and during the growing season in
August. Lateral solute concentration gradients were assessed by
sampling surface water (0-3h from start of sampling), followed
immediately by in-channel pore water (3-8 h) and then outof-channel groundwater (6-34 h). MINIPOINT sampling was
conducted during the May assessment.

Stream flow Data Collection

Instantaneous stream discharge was measured during
surface water sampling at a location immediately downstream of
the study reach using acoustic Doppler velocimetry (ADV) [22].
Surface-water samples were collected at the mid-time of three
consecutive discharge measurements (1 h each). Subsequently,
paired Lagrangian discharge measurements were made in
triplicate (one h intervals), with upstream and downstream
measurements offset by the average downstream transport time
(25 and 40 min) through the reach. Surface-water transport time
was calculated using the average stream flow velocity measured
immediately downstream of the reach before sample collection.

Water-Quality Data

For each assessment, triplicate surface-water samples were
collected at upstream and downstream boundaries using a
Lagrangian sampling approach [13]. Surface-water was collected
at one hour intervals at the upstream margin without entering
the stream, using a pole to position sample bottles at mid depth in
the center of flow until full (Figure S2 in File S1). Corresponding
downstream samples were collected following a delay (25 and
40 min) equivalent to the average surface-water transport time
through the reach. Ultra-trace-level clean-sampling, processing, and Hg analysis procedures were as in [6,9, 30, 31]. Filtered (0.7
μm quartz-fiber filter) samples were analyzed for FMeHg by gas
chromatographic separation with cold vapor atomic fluorescence
spectrometry [32] and for FTHg by oxidation, purge and trap,
and cold vapor atomic fluorescence spectrometry [33] at the
USGS Mercury Research Laboratory (Middleton, Wisconsin).
The reporting limit for FMeHg and FTHg was 0.04 ng/L.
Concentrations of DOC [34]and chloride [35]were determined as
cited.

Vertical hydraulic gradients in the stream channel at the
time of sample collection were estimated as the potentiometric
difference between hyporheic pore water (water level inside
in-channel piezometers) and surface water (water level
outside the piezometers) using an electric water level tape.
Porewater samples were collected from in-channel piezometers
using a peristaltic pump (flow less than 50 mL min-1) and low
volume (200 mL purge; 1 L total pumped volume) sampling
techniques [13]. An assessment of water quality parameters (pH,
conductivity, and temperature) over time was conducted to verify
stream-water entrainment did not occur under these pumping
conditions [13]. Out-of-channel groundwater wells were purged
(3 well volumes) on day 1, immediately after water-elevation
measurements. Groundwater was sampled and processed as
described for in-channel porewater samples. Ultra-low-flow
(approximately 2 mL min-1) MINIPOINT sampling was conducted
[as described in detail elsewhere 28] to collect porewater from
the shallow hyporheic-flow and underlying groundwaterhyporheic
porewater mixing zones using a peristaltic pump.
All samples were filtered and preserved with 6.5 N trace-metal
grade HCl in the field in a temporary glove box[13](Figure S3 in
File S1), stored on ice, and shipped overnight to the lab. Fresh
gloves, glove-box bags, and pre-cleaned filter assemblies were
used for each sample. Peristaltic tubing was acid cleaned (ultrapure
5% HCl) and rinsed (distilled water) between samples [6,
13, 31]. Additional sampling details provided in File S1.

Statistical significance metrics and percentage reach
flux increases provided direct, quantitative measures of the
importance of reach wetlands as sources of water and solutes to
the aquatic habitat during each 3 h surface-water flux assessment.
Reach discharge results were then combined with hyporheic
zone hydraulic gradient data to assess the relative importance of
advective and diffusive wetland-stream solute exchange. Solute
concentration gradients (e.g., groundwater to surface water,
in-channel porewater to surface water) clarified the effect of groundwater discharge (dilution or concentration of solutes)
on stream solute concentrations and provided insight into the
direction and magnitude of diffusive exchange in the event that
groundwater discharge was insignificant.

The One-tailed paired t-test was employed to assess flux
differences between the top and bottom of the study reach.
Kruskal-Wallis ANOVA on Ranks was combined with Dunn's
Multiple Comparison Method to identify significant differences
between four lateral gradient groups: groundwater, porewater
at the channel margin, porewater at the center of channel,
and stream surface water. A significance level (α) of 0.05 was
employed for all comparisons.

Results and Discussion

The demonstrated importance of wetland areas as sources
of MeHg and THg to the stream and the substantial temporal
variability in MeHg supply to the stream during 2007-2008
(Figure 2;[7] )indicated that Sixmile Brook was a suitable location
to evaluate wetland-stream hydraulic and chemical gradients as
controls on short-term wetland-stream solute transport under
different hydrologic and geochemical conditions. These short
(25 and 40min per Lagrangian sample pair, approximately 3
h total) reach synoptics were intended to rigorously assess
"instantaneous" gradients and wetland-stream fluxes and
were not intended to represent longer-term wetland-stream
dynamics[assessed previously in 7].

Change in Sixmile Brook Reach Fluxes

During May 2009 snowmelt event, a significant (p<0.005)
increase in stream discharge of 7.1±1.3 % was observed
within the Sixmile Brook study reach (Table 1). Because no
tributaries existed within the reach, increases in discharge
between upstream and downstream measurements were
attributed to groundwater fluxes. Comparable (8.0±1.3% and
9.0±1.3%) significant (p<0.005) increases in the fluxes of DOC
and dissolved chloride (respectively) were also observed within
the reach, demonstrating efficient hydrologic transport of
dissolved constituents from wetland sediments to the stream
habitat. However, no statistically significant (p>0.16) changes
in the fluxes of FMeHg or FTHg were observed, demonstrating
that discharging groundwater was not a significant source of
Hg within the study reach during this short surface-water flux
assessment. Because significant increases in fluxes of water, DOC,
and chloride within the 25 min downstream travel time indicated
good wetland-stream hydrologic connectivity and a favorable
hydraulic gradient, the lack of significant net fluxes of FMeHg and
FTHg was hypothesized to be due to dilute Hg concentrations in
groundwater discharge within the study reach during the May
assessment.

During the August 2009 growing-season assessment, no
statistically significant changes in discharge or in the fluxes of
FMeHg, FTHg, DOC or chloride were observed in the Sixmile
Brook study reach, indicating that the adjacent riparian wetlands
were not a significant source to the reach aquatic habitat
during the 40 min downstream travel time(Table 1).During this sampling event, however, the apparent lack of net exchange of
Hg and other solutes was attributed to insignificant advective
transport between shallow groundwater and the stream. Because
the results of the May flux assessment demonstrated good
wetland-stream hydrologic connectivity within the reach, the
lack of net groundwater discharge during the August event was
hypothesized to be due to insignificant wetland-stream hydraulic
gradient. In the absence of substantial net hydrologic transport to
the stream, solute exchange would be limited to hyporheic zone
mixing and diffusive processes at the sediment/surface-water
interface, with minimal contribution to reach fluvial chemistry
during the approximate 40 minute surface-water travel time
between upstream and downstream reach boundaries. The lack
of significant change in DOC and dissolved chloride in the absence
of an increase in discharge corroborates the previous conclusion
that DOC and chloride flux increases observed in May 2009 were
attributable to groundwater discharge within the Sixmile reach.

The Sixmile study reach represented less than 4% of the total
upstream basin area[20].The influx of upstream surface-water
was the primary (generally greater than 90%) source of water
and solutes (FMeHg, FTHg, DOC, and chloride) to the study reach
for both flux assessment events. The mean percentage increases
(7-9%) in fluxes of surface water, DOC, and chloride observed
in the study reach in May 2009, however, were approximately
two times greater than the corresponding increase in basin
area, indicating that the riparian wetlands within the reach were
important sources of water and solutes to the stream habitat. In
August 2009, the flux of water and solutes at the downstream
margin of the study reach was entirely attributable to surfacewater
influx at the upstream margin, demonstrating that
contributions from discrete wetland areas vary substantially
over time. There was no evidence of significant supply of FMeHg
or FTHg from the adjacent wetlands to the aquatic habitat of the
Sixmile Brook study reach (Table 1) during either flux assessment,
despite the spatial extent of the riparian wetlands bordering
the reach (Figure 1) and the potential for solute exchange (DOC
and chloride) demonstrated during May (Table 1, Figure 3A-
3D).The elevated concentrations of MeHg observed in several
wetland piezometers (compared with hyporheic piezometers
at channel margins, see Figures 3A and [7] ) and the observed
fluvial transport of MeHg from wetland-dominated reaches upstream in Sixmile Brook are consistent with wetland areas as
primary sources of MeHg to the stream throughout the growing
season (Figures 2A and 3A,[7] ). The current results verify the
substantial temporal variability in wetland-stream MeHg supply
reported previously (Figure 2) [7] and indicate the importance of
wetland-stream hydraulic and chemical gradients as important
contributors to temporal variability.

The Sixmile Brook flux results are in marked contrast to
the consistent, significant (p < 0.0001) increases (generally 10
% or more) in both water and dissolved constituents (FMeHg,
FTHg, DOC, chloride) previously reported in sister study area at
McTier Creek during two 2009 sampling events(Table1; [13]).
To better understand the comparative lack of net FMeHg and
FTHg exchange between the adjacent riparian wetlands and the
aquatic habitat within the Sixmile Brook study reach during the
2009, surface-water flux assessments, geochemical gradients
between the wetland and the stream channel, as well as stream
channel vertical hydraulic gradients were assessed.

Groundwater to Surface Water Gradients

Differences in solute concentrations between surface
water samples and samples of groundwater collected from
wetland piezometers (located 3-10 m from the stream bank)
were evaluated to assess the potential importance of shallow
wetland groundwater as a longer-term source of solutes to the
stream. Results from piezometers installed across the stream
channel at the upstream and downstream reach boundaries
were used to assess the potential importance of groundwater
discharge as a proximal source of solutes to the stream at the
time of sample collection. Piezometers emplaced at the channel
margins provided insight into the importance of shallow wetland
groundwater discharge to the stream, while piezometers placed
near the center of the channel reflected the contribution of
comparatively deeper groundwater [13].

During May 2009, net positive (toward surface water
compartment) vertical hydraulic gradients ranging from 0.025-
0.05 (0.5-1.0 cm water level change over 20 cm distance; data
not shown) were observed in piezometers at the channel
margins of Sixmile Brook, consistent with ongoing discharge of
shallow wetland groundwater to the stream and the observed

significant increase in surface-water flux within the reach. No
vertical hydraulic gradients were detectable (potential difference
less than 2 mm) in piezometers located in the center of the
channel. Concentrations of DOC and chloride in channel margin
piezometers were comparable to those observed in surface water,
consistent with the significant increases in DOC and chloride
fluxes within the study reach (Figure 3C &3D). In contrast, FMeHg
concentrations (Figure 3A) in channel margin piezometers
were significantly (t-test; p < 0.0001) less than in surface water
(mean concentration approximately 15% of mean surface water
concentration), consistent with the lack of detectable change
in FMeHg flux within the reach (Table 1). Similarly, FTHg
concentrations (Figure 3B) in channel margin piezometers were
significantly (t‑test; p = 0.003) less than in surface water (mean
channel margin concentration approximately half of mean surface
water concentration), consistent with the lack of significant
change in FTHg flux within the reach during the May event
(Table 1). It is noteworthy that surface-water FMeHg and FTHg
concentrations were within the range (not statistically different;
Kruskal-Wallis One-Way ANOVA on ranks; p > 0.05) observed
in groundwater piezometers located 3-10 m from the stream
channel (Figure 3A &3B), indicating that shallow groundwater
was an important source of FMeHg and FTHg to the stream over
longer time periods. However, the low concentrations of FMeHg
and FTHg observed in channel margin piezometers indicated
that dilute-Hg groundwater was discharging to the stream reach
during the May stream-water flux assessment.

No vertical hydraulic gradients were detected in stream
piezometers (margin or center of channel) during August 2009
(data not shown), suggesting minimal groundwater discharge
and consistent with the lack of significant change in the fluxes
of water, FMeHg, FTHg, DOC, or chloride within the Sixmile
Brook reach. No significant differences in DOC concentrations
were observed between groundwater, hyporheic porewater,
and surface water (Figure 3G), indicating the potential for
groundwater as a substantial source of DOC to the stream
under favorable hydrologic conditions. Combined with the
lack of significant change in discharge within the reach, this
result demonstrates that the lack of increased DOC flux within
the study reach was due to little or no hydraulic gradient and
a corresponding lack of advective hydrologic transport from
wetlands to the stream. In contrast, concentrations of FMeHg,
FTHg, and chloride were statistically significantly higher in
surface water than in groundwater or porewater. The lack
of significant changes in fluxes within the reach, the lack of
detectable vertical hydraulic gradients, and lower concentrations
in groundwater than surface water indicated that the extensive
riparian wetlands within the reach were not a significant source
of Hg to the stream reach during the short August 2009 streamwater
flux assessment period.

In contrast to Sixmile Brook, concentrations of FMeHg,
FTHg, DOC, and chloride in groundwater and in-channel pore
water piezometer samples were comparable to or greater than
observed in surface-water samples at McTier Creek (Figure 4A-
4D), consistent with groundwater as a substantial source of these
solutes to the stream habitat under the favorable hydrologic

conditions observed in this study. Mass balance calculations and
water-stable-isotope mixing results indicated that 80% or more
of the observed increases in discharge and solute fluxes within
the McTier Creek study reach were attributable to groundwater
discharge [13].

Hyporheic Vertical Profiles

MINIPOINT vertical concentration profiles obtained from
the margin and center of the Sixmile Brook stream channel

Figure 4:Concentrations of filtered methylmercury (A), filtered total
mercury (B), dissolved organic carbon (C), and dissolved chloride (D)
at McTier Creek (April and July data did not differ significantly and are
combined) in 2009. Other labeling as in Figure 3.

during May provided additional insight into the supply of
solutes to the stream reach (Figure 5A-D). Because MINIPOINT
assessments were conducted once and thus lacked estimates
of sample variability, these concentration profiles were used
only to 1)assess centimeter-scale trends in vertical hyporheic
concentration gradients, 2) identify the depth of the surface-water
mixing (hyporheic flow) zone, and 3) verify relative differences
in chemical signatures in shallow groundwater samples collected
at the channel margin and comparatively deeper groundwater
samples collected from the channel center.

Chloride profiles indicated a well-mixed hyporheic-flow
porewater layer (indistinguishable from surface water to a
depth of approximately 20 cm below the sediment/surface
water interface) and decreasing concentrations with increasing
depth from 20-60 cm (Figure 5D). FMeHg, FTHg, and DOC
concentrations also generally decreased with increasing depth,
consistent with the previous conclusion that the primary source
of these solutes to the stream reach was upstream surface
water. The fact that channel margin, porewater FMeHg and
FTHg concentrations below the surface mixed (hyporheic flow)
layer were approximately an order of magnitude less than in the
overlying surface water is consistent with the discharge of deeper groundwater with dilute Hg and the lack of significant change in
FMeHg and FTHg fluxes within the study reach during the May
event. The fact that concentration profiles from the margin and
center of the channel were comparable for the conservative
chloride ion (Figure 5D) as well as for FTHg and DOC (Figure 4AC)
is also consistent with surface water as the primary control on
these shallow, hyporheic-porewater concentrations during the
May flux assessment. However, concentrations of FMeHg in the
hyporheic (0-20 cm) zone were generally higher at the channel
margin than at the channel center (Figure 5A), consistent with
previous conclusions that wetlands are longer-term sources of
FMeHg to Sixmile Brook [7].

In contrast, only limited changes in concentrations with
depth were observed in MINIPOINT samples from McTier Creek
in July 2009, and solute concentrations in porewater and surfacewater
samples were comparable (Figure 5E-H). These results are
in agreement with the documented importance of groundwater
discharge in the McTier Creek study reach [13] under nonflood
conditions. FMeHg, FTHg, and DOC concentrations were
consistently higher at the channel margin than at the center of
channel, further supporting the conclusion that discharge of
shallow groundwater from adjacent wetland/riparian floodplain
areas is the primary source of these constituents to the stream.
In contrast, chloride concentrations were consistently higher
at the center channel location than at the channel margin and
comparable to surface-water concentrations, indicating that the
primary sources of chloride in the reach were surface water from
upstream of the reach and discharge of deeper groundwater
within the reach. The fact that surface-water concentrations
of FMeHg, FTHg, and DOC were intermediate to MINIPOINT
concentrations from the margin and center locations (Figure 5E-
5G) is consistent with the single-screen in-channel Piezometer
results (Figure 4A-4C) and indicates that surface water in the
stream reach is a mixture of shallow and comparatively deeper
groundwater discharge.

Implications for MeHg Supply

The results of this study verify that the extensive riparian
wetlands that border Sixmile Brook are hydrologically well
connected to the stream. Consequently, the results are compatible
with the earlier conclusion that adjacent and upstream wetland
areas are important long-term sources of water and solutes
to the Sixmile Brook aquatic environment [7]. These results
also support the hypothesis that unfavorable instantaneous
wetland-stream hydraulic and chemical gradients (direction
and magnitude) limit the importance of these wetland areas as
sources of Hg and other solutes to Adirondacks-region stream
habitats in the short term.

Hg-risk management strategies are applied at regional to
national scales, while foundational research on environmental
Hg processes and dynamics is most often conducted at
focused-reach to small-watershed scales. The development of
mechanistic models that extend research-scale findings to Hgrisk-
management scales is challenged by competing needs to
capture those Hg processes which are most essential to system
simulation while maintaining model usability (i.e., avoiding
parameter inflation and excessive processing time). Numerical
model improvements that allow more accurate prediction of
temporal and spatial variations in fluvial MeHg supply are needed
to better manage Hg-bioaccumulation risk in the Adirondacks
and other regions of North America [19, 36]. The current
results suggest that continued investigation of factors that affect
temporal variations in wetland-stream gradients and improved
near-stream hydrologic models may substantially improve Hgrisk-
management models.

Acknowledgements

The U.S. Geological Survey National Water Quality Assessment and Toxic Substances Hydrology Programs supported this
research. We thank Finch-Pruyn and the Nature Conservancy as
well as the family of Senator Strom Thurmond for access to the
SixmileBrook and McTier Creek study reaches, respectively.

Data Availability

Data in Figures 2-4 can be downloaded from the USGS
National Water Information System (NWIS) at (http://dx.doi.
org/10.5066/F7P55KJN). Data in Figure 5 are available at
(http://dx.doi.org/10.5066/F7QN64TC).